I. Field of the Invention
This invention relates to a method for electroporation for introducing a gene, virus or an organelle into a cell.
II. Description of the Related Art
A method in which protoplasts and DNAs are suspended in a buffer and applying an electric voltage ("electro") across the suspension so as to form repairable small pores (poration) into the cell membrane of the protoplasts, thereby carrying out the introduction of the DNAs into the protoplast cells, is called electroporation. This method was developed recently and is becoming widely employed. In fact, transformants were obtained using this method in a number of plants such as tobacco and rice. In the conventional electroporation method, buffers containing a high concentration of chloride such as potassium chloride, sodium chloride, calcium chloride and magnesium chloride are often used (Fromm et al., Nature 319:791-793, (1986); Shimamoto et al., Nature 338:274-276 (1989); and Dekeser et al., Plant Physiol. 90:217-223 (1989)). Potassium ion is contained in plant cells at high concentration, and so the potassium ion contained in the buffer is thought to be required for compensating the potassium ion flowing out from the pores in the cell membrane. Further, calcium ion and magnesium ion are thought to contribute to the stabilization of the cell membrane, so that these ions in the buffer are assumed to be utilized for stabilization.
However, with the conventional electroporation process, the cells are more or less damaged, so that the frequency of the cell division of the cells is reduced. As a result, the efficiency of the transformation by the electroporation is not satisfactorily high.
One of the causes of the reduction in the cell division frequency is the damage of the cells directly given by the application of the high voltage. This drawback is thought to be inherent to the electroporation itself and may not be overcome. However, it is thought that this is not the only cause of the reduction in the cell division frequency. Thus, it is expected that the frequency of the cell division may be considerably increased by eliminating such other causes.